Full text of "USPTO Patents Application 10687850"

See other formats

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification * :
C12Q 1/00

(11) Internationa! Publication Number: WO 96/94398

(43) Internationa) Publication Date: 15 February 1996 (15.02.96)

(21) Internationa) Application Number: PCT/GB95/01818

(22) International Filing Date: 1 August 1995 (01.08.95)

(30) Priority Data:
9415499.4

1 August 1994 (01.08.94)

(71) Applicant (for ail designated States except US)i MEDI SENSE

INC [US/US); 266 Second Avenue, Waltham, MA 02154
(US).

(72) Inventors; and

(75) Inventors/Applicants (for US onfy): SANGHERA, Gurdial,
Singh [GB7GB]; 3 Kennett Road, Hoodington, Oxford OX3
7BH (GB). BARTLETT, Philip, Nigel [GB/GB]; University
of Southampton, Dept of Chemistry, Southampton S09
5NH (GB). BIRKIN, Peter, Robert (GB/GB]; Univexsrty of
Southampton, Dept of Chemistry, Southampton S09 5NH
(GB).

(74) Agent: HITCHCOCK, Esmond, A4 Uoyd Wise Trcgear & Co.,
Norman House, 105-109 Strand, London WC2R 0AE (GB).

(81) Designated States: AM, AT. AU, BB, BG, BR. BY, CA, CH,
CN, CZ, DE, DK, EE, ES. FL GB, GE. HU, JP, KE, KG.
KP. KR, KZ, LK, LR, LT. LU, LV. MD. MG. MN, MW,
MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG. SI, SK, TJ,
TT, UA. UG, US, UZ, VN, European patent (AT, BE, CH.
DE. DK, ES, FR. GB. GR, IE, IT, LU, MC, NL, PT, SE),
OAPI patent (BF, BJ. CF, CG, CI, CM, GA, GN, ML, MR.
NE, SN, TD, TG), ARIPO patent (KE, MW, SD, SZ, UG).

Published

With international search report.

Before the expiration of the time limit for amending the
claims and to be republished in the evens of the receipt of
amendments.

(54) Title: ELECTRODES AND THEIR USE IN ANALYSIS
(57) Abstract

A method for indicating the concentration of a substance in solution comprises passing an alternating voltage between a first electrode
structure having coated thereon a polymer arid a second separate counter electrode in the solution. The polymer is in one of an oxidised
and a reduced state, between which states its conductivity varies. Changes in the conductivity of the polymer coating are measured, the
measurement being representative of the state of the polymer and thereby of the concentration of the substance in the solution. Electrodes
for use in this method are also described. *

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify Stales party to the PCT on the front pages of pamphlets publishing international
applications under the PCT.

Austria

Australia

Barbados

Burkina Faso

Bulgaria

Bean

Brazil

Betas

Canada

Centra] African Republic

Congo

Switzerland

Cote d*I voire

Canwjooo

China

Chechoslovakia

Czech Repubbc

Germany

Denmark

Spain

Finland

France

Gabon

Unncd Kingdom

Georgia

Guinea

Greece

Hungary

Ireland

Italy

Japan

Kenya

Kyrgyatan

Democratic People'* Republic

of Korea

RepubSc of Korea

Kazakhstan

Sri Lanka

Lpaembourg

Latvia

Monaco

Repubbc of Moldova

Madagascar

Mali

Mongolia

Mauritania

Malawi

Niger

Netherlands *

Norway

New Zealand

Poland

Portugal

Romania

Russian Federation

Sudan

Sweden

Slovenia

Slovakia

Senegal

Chad

Togo

Tajikistan

Trinidad and Tobago

Ukraine

Unfied States of Ame

Uzbekistan

Viet Nam

WO96/0439*

PCT/GB95/01818

ELECTRODES AND THEIR USE IN ANALYSIS

This invention relates to electrochemistry and in
particular to novel electrode structures and their use in
analysis for the detection of enzymes or their substrates.
The invention is of particular utility in sensors for
biomedical applications.

There is a continuing need for improved immunoassay *
techniques particularly for the detection of low levels of
analytes in small samples.

In recent years, a number of electrochemical techniques
have been developed in the hope of avoiding the generally
conventional use of radioisotope labels for the detection of
low levels of analytes. Direct amperometric measurements of
NADH and of phenol has been reported by Wright fit al Anal.
chem > * 58 (1986) 2995 and by Wehmeyer fit al Anal. Chem. .
58(1986) 135 employing enzyme -labelled antigens. The use of
redox-labelled drug conjugates, specifically ferrocenyl-
lidocaine for similar direct amperometric measurements has
also been reported by Di Gleria et al Anal. chem. 58(1986)
1203. These direct amperometric measurement techniques tend
to suffer from a sensitivity limitation and in general the
techniques are limited to low micromolar concentration
ranges .

Chambers and Walton in J. Electroanal rhom , 250(1988)
417-425 report use of a poly (vinylf errocene) -modified glassy
carbon electrode used as a charge -accumulating device to a
accumulate charge from a solution of glucose/glucose oxidase
via the intermediary of a redox-labelled antigen, which is
cycled repeatedly between the enzyme and the electrode.
Chambers and Walton were able reproducibly to detect 1,1'-
dimethylferrocene-3-ethan-l-ol-2-amine at nanomolar

WO 96/04398

PCT/GB95/01818

concentrations. Although Chambers and Walton identified 8-
ferrocenyl- theophylline as their preferred redox-active
mediator for further work, they found that it possessed a
significant disadvantage in that it appeared to be bound
unspecif ically to anti- theophylline antibodies and so was
not ideal for use in a competitive immunoassay.

A different approach has been adopted by Bart let t and
Birkin, ( Anal. Chem . 1993, 65, 1118-1119), namely the
production of a DC bioelectrochemical transistor in the form
of an enzyme switch. In this prior enzyme switch system, to
provide a switch responsive to glucose, glucose oxidase was
immobilized in a thin insulating film of poly (1,2-
diaminobenzene) deposited on top of a poly (aniline) film
deposited across a gap between two carbon electrodes. The
change in conductivity of poly (aniline) between its oxidised
and reduced states is measured by applying a small DC
voltage from one carbon electrode to the other across the
film and is used to measure the presence of redox mediator
shuttling charge between the enzyme and polymer film. One
drawback of this system is the need to form a polymer film
which bridges the two electrodes, taking into account the
minimum distance which can be tolerated between the
electrodes.

The present invention has arisen from the work of the
present inventors in seeking to improve upon the prior
techniques .

As will be seen from the detailed description below,
the methods and apparatus of the present invention draw upon
the technology developed in connection with the prior enzyme
switches, but apply this technology in a quite new way,
namely in an alternating current system using a single
polymer coated electrode.

In accordance with a first aspect of the present

WO 96/04398

PCT/GB95/018J8

invention, there is provided a method for indicating the
concentration of a substance in solution comprising passing
an alternating voltage between a first electrode structure
having coated thereon a polymer, the polymer being in one of
an oxidised and a reduced state between which states its
conductivity varies, and a second separate counter electrode
in said solution; and measuring the change in conductivity
of the polymer coating, such measurement being
representative of the state of the polymer and thereby of
the concentration of said substance in the solution.

For use in the above method the invention also provides
an electrode structure forming a single electrode in a
circuit comprising an electrode having coated thereon a
polymer film the polymer being in one of an oxidised and a
reduced state between which states its conductivity varies.

In a preferred embodiment of the invention, the method
is used to provide an indication of the concentration of an
enzyme substrate in solution. The enzyme is preferably
immobilised in a film overlying the polymer coating and is
involved in redox reactions with its substrate present in
the solution. As the enzyme /substrate system undergoes
redox reaction, charge is transferred to or from the polymer
film bringing about a change in the oxidation state of .the
polymer. This change in oxidation state leads to a
variation in the conductivity of. the polymer. The rate at
which this change occurs is dependent on the concentration
of the substrate in solution so that by measuring the
impedance of the system over time, the concentration of the
substrate may be determined. Alternatively, a substrate may
be immobilised on the polymer coating and the electrode is
used to indicate the concentration of an enzyme in solution.

While in preferred embodiments, the electrode finds use
in an enzyme system, we have also found that the method
allows for direct measurement of substrates which undergo

WO 96/04598

PCT/GB95/01818

redox reactions. For example, NADH and L-ascorbic acid have
been oxidised in the presence of a polymer film which
undergoes reduction and thus changes from an insulating to
a conducting state. This changes the conductivity of the
film as outlined above to measure directly the concentration
of a particular substance in solution. Alternatively, the
system may be used to identify changes in the redox
condition of the polymer film which are not a direct result
of an enzyme/substrate system i.e. the background activity
action, to provide a more accurate indication of the enzyme
or substrate concentration.

Redox mediators may also be provided in the solution or
immobilised on the support to transfer charge to or from the
polymer film. As the substance or the substrate in the
presence of the enzyme undergoes reduction or oxidation, the
polymer support will also undergo oxidation or reduction
either directly or through the use of mediators.

The method may also be carried out in the absence of
the substance whose concentration is to be determined either
with the enzyme electrode or base polymer film electrode to
monitor background charges in the polymer conductivity which
are not related to the concentration of the substance of
interest .

The invention is described in detail with particular
reference to the glucose/glucose oxidase system, employing
poly (aniline) as the conductive polymer. The invention is
not so limited but may be applied broadly to other
enzyme/ substrate systems and may also be used with other
polymers, with changing conductivity depending on their redox
state.

The use of a single electrode associated with a polymer
film allows for smaller electrodes to be produced having
thinner coatings of polymer film. This has the advantage in

WO 96/04198

PCT/GB95/01818

that it is not necessary to form the film as a gap between
two electrodes which may be difficult due to limitations in
the minimum size gap that can be formed or tolerated without
short-circuiting. Furthermore, the system is more sensitive
when smaller quantities of polymer are used. Since the
change in oxidation state of the polymer has a direct effect
on conductivity, if less charge transfer is required to
switch the polymer film from an insulating to a conducting
state, the system becomes more sensitive to smaller
quantities of substrate.

The electrode structure of the invention can be in the
form of a micro electrode which enables commercial
application of the technique as a bid-sensor where very
small sample volume are involved, such as 10 /xl. Analyte
may be present in micromolar concentrations or less. The
sensitivity of the system may allow concentrations in the
femtomolar range to be detected, particularly where polymer
films of thickness in the range O.litm to 1/xra are applied to
the electrode.

The invention has been described with reference to
poly (aniline) which is insulating in its oxidised state and
conducting in a reduced state. It may be readily returned
to its oxidised state by holding the potential of the
electrode at +0.4V vs. saturated calomel electrode (SCE) in
a clean pH5 buffer solution.

The invention is hereinafter described in more detail
by way of example only with reference to the accompanying
drawings in which: -

Fig. 1 is a drawing illustrating the experimental set
up for use of an electrode structure in accordance with the
present invention;

Fig. 2 is a circuit diagram of the system of Fig. 1;

WO 96/04398

PCT/GB95/01818

Fig. 3 is an exploded view of a carbon microband
electrode used in the present application;

Fig. 4 is a greatly enlarged schematic representation
of the cross section of an electrode of the present
application;

Fig. 5 is a schematic diagram of a reaction scheme of
the present application;

Fig. 6 is a schematic diagram of an alternative
reaction scheme;

Fig. 7 shows measurements of impedance against time for
one example of electrode structure in accordance with the
present invention when exposed to different concentrations
of glucose in solution;

Fig. 8 shows how the glucose concentration is related
to the switching rate (defined as the transient maximum
gradient divided by the impedance change over the time
period recorded) as derived from Fig. 7 for the electrode
structure concerned;

Fig. 9 shows measurement of impedance against time for
an example of electrode structure in accordance with the
present invention when exposed to difference concentrations
of L-ascorbic acid in solution;

Fig. 10 shows how the ascorbic acid concentration is
related to the switching rate as derived from Fig. 9 for the
electrode structure concerned; and

Fig. 11 is a schematic representation of the cross
section of an alternative electrode of the present
application.

WO 96/04398

PCT/GB95/0J818

The method of the present application involves forming
a circuit comprising an electrode having coated thereon a
polymer film and a counter electrode, an AC voltage being
passed through the circuit to establish the impedance of the
system between the electrodes. The conductance of the
polymer film alters depending on the oxidation state of the
polymer, which is in turn altered in response to oxidization
or reduction reactions occurring to substrates in the
solution in which the electrodes are placed.

Representative circuit diagrams are shown in Figs. 1
and 2. The polymer coated electrode (1 of Fig. i) i s
represented by the equivalent circuit of a resistance Rp
which ranges from 50 ohms to 100 kiloohms and a capacitance
Cp in parallel. A blocking capacitor C b may be mounted -in
series with the electrode to prevent any unwanted DC signal
affecting the oxidation state of the polymer. The
resistance Ru is uncompensated and represents the resistance
of the solution (2) and of the counter electrode (3) which
may suitably be formed of platinum gauze. An alternating
voltage V ac is connected across the counter electrode and the
blocking capacitor. The change in conductivity of the
polymer film is readily detectable and the rate at which the
film is switched from an insulating state to a conducting
state (or vice versa) is dependent upon the concentration of
the substrate in the solution which is undergoing redox
reaction, 4

In its simplest form, a polymer film is formed over a
carbon electrode. A suitable polymer is poly (aniline) which
is insulating in its oxidised state and conducting in a
reduced state. The change in the oxidation state of the
poly (aniline) film is brought about by redox reactions
occurring in the solution and/or at the polymer/solution
interface. Mediators may be added to shuttle charge from
the redox reactions occurring in the solution and the
polymer film. An example of the reaction in its simplest

WO 96/04398

PCT/GB95/01818

form is shown at Fig. 6 where the oxidation of L- ascorbic
acid leads to direct oxidation products while at the same
time the poly (aniline) film is reduced from an insulating to
conducting state. Similar results may be seen with NADH.

In an alternative aspect of the invention, the
electrode is used in an enzyme system to measure the
concentration of a substrate. In a preferred embodiment,
the enzyme is immobilized in a thin insulating film on the
polymer film. Such a system is the glucose/glucoseoxidase
system exemplified by Fig. 5 where oxidation of glucose
leads to reduction of the glucose oxidase enzyme. A
mediator may be included such as tetrathiafulvalene or
f errocyanate [Fe (CN) J 3 " .

While it is not essential for the enzyme to be bound in
a separate layer over the polymer film, it is preferable.
Binding the enzyme to the polymer electrode allows smaller
quantities of enzyme to be used and aids ease of handling
the device, and thus increases the reproducibility of
results obtained with the device.

In a preferred aspect of the invention, the polymer
electrode is formed as a number of carbon microband gap
electrodes on which a polymer film is applied. These
microband electrodes may be produced by screen printing onto
a PVC card and are shown schematically in Fig. 3.
Typically, each card may have a number carbon microband gap
electrodes formed thereon which are subsequently separated.
Firstly, a carbon electrode pattern (4) is printed onto a
PVC card (5) . Subsequently two layers of dielectric (6) are
printed followed by a second carbon electrode layer (7)
which lies directly over the first carbon electrode layer
(4) . Two more layers of dielectric (8) are applied to this
second electrode layer (7). After each printing step, the
PVC card is placed in an oven at 55°C until the freshly
applied layer has dried. Typically, this takes about 30 to

WO 96/04398

PCT/GB95/D1818

60 minutes. The carbon layers are determined to be dry when
the resistance of the carbon track (measured with a Digital
Volt Meter) reached a minimum constant value of typically of
about one kiloohm. Fig, 3 shows an exploded view of a
carbon microband single electrode having two carbon layers.
A single carbon layer could also be used.

To expose the microbancT electrodes, the finished
structures are sheared across the two carbon layers to
expose the edges of the two carbon print layers. This is
achieved by freezing the electrodes in liquid nitrogen and
then fracturing them to avoid deformation and problems with
shorting which can occur if the electrodes are cut
mechanically to expose the carbon microband. The carbon
microband electrodes are approximately 4.5 millimetres in
length and 10 to 15 pm wide separated by a 20 fim gap,

A layer of poly (aniline) (PANI) is then grown on to the
carbon microband electrode by holding the potential of the
carbon electrode for 20 seconds at +0.9 V vs. SCE in a
solution containing 200 /xL of aniline and 5 cm 3 of NaHS0 4
acidified to a pH of approximately 0 with 0.5 cm 3 of
concentrated H 2 SO«, suitably at 95-98% AR grade. Each of the
above reagents may be obtained from Aldrich Chemical Co.
Gillingham, Dorset. The typical total charge passed during
this deposition was 3mC, and corresponds to a film thickness
in the region of 20jzm.

Fig. 4 shows a schematic view of the electrode, having
two carbon microband electrodes (4,7) having a poly (aniline)
film (9) applied thereto. An enzyme (10) such as glucose
oxidase is immobilised in an insulating film (11) applied to
the surface of the poly (aniline) film (9) . In the method of
the invention, this electrode structure is used as a single
electrode.

While the electrode described above has a 20^m polymer

WO 96/04398

PCT/GB95/018I8

film and a 20/im gap, single electrodes for use in the
present invention may be fabricated with significantly
thinner coatings of polymer in the region of 0.1 to 1 /im
enabling a wider range of polymer films to be applied and
enhanced sensitivity.

Though it has been found that the use of poly (aniline)
provides good results, this conductive polymer is pH
dependent. The invention is not limited to the use of this
specific polymer but can employ any other polymer which has
an oxidized and a reduced state and is electrically
conductive in one of these states.

Among the wide variety of electrically conductive
polymers usable in practice of the present invention for
appropriate systems are:

Poly (aniline) and derivatives thereof;

Poly (thiophene) and derivatives thereof ;

Poly (pyrrole) and derivatives thereof; and

Poly (pyridine) and derivatives thereof.

Reference may be made to the specification of
International Patent Application PCT/US88/02319 published
under No: WO 89/01694 of Allied Signal, Inc which describes
the manufacture and use of thermally stable forms of
electrically conductive polyaniline. The polyaniline is
rendered conductive by the use of a dopant solute which upon
addition to the polyaniline ionizes the polymer with
concomitant formation of an ionic dopant solute species. In
general, suitable compounds for addition to the polymer have
the formula;

[R - SO,') n .M' n .

where:

R is an organic radical;

M* n is a species having a positive charge equal to n, n
being 1 to 4. In the preferred arrangement M is univalent
and preferably hydrogen.

WO 96/04398

PCT/GB95/01818

Similar such dopants may be incorporated in the
poly (thiophene) , poly (pyrrole) , poly(indole) and
poly (pyridine) systems. A wide variety of suitable monomers
may be used for forming the polymers. The selection of
individual monomers will have an effect on the film-forming
properties as will the length of the polymer backbone, as
will be well understood by polymer chemists and would be a
matter of appropriate adjustment to provide a polymer of
desired physical properties. ; By way of example, typical
monomers are set out below for each of the abovenoted
polymer systems:

Poly (aniline)

(m + n) is no greater than 5

NR r

Poly (pyrrole)

Poly (thiophene)

H A s iL H

WO 96/04398

PCT/GB95/01818

Poly (indole)

(m + n) is no greater than 4
Poly (pyridine)

(m + n) is no greater than 5

R, Ri# X>2r R3 and R 4 are suitable organic radicals the
selection of which will be well understood by polymer
chemists. Most preferably, each of R, R lf R 2 , R 3 and R 4 will
be lower alkyl (i.e., 1 to 4) .

There is a substantial ease in detection by connecting
the single electrode in a simple alternating current
circuit. From the above, it is clear that by reference to
a single electrode, a microband electrode structure may be
used. By virtue of the presence of the counter electrode
and the construction of the device, it is in effect a single
electrode. No special reference electrode is required. Any
appropriate counter electrode may be employed to complete
the circuit since its resistance is in effect subsumed
within the uncompensated resistance R^ and what is being
detected is the change in conductivity of the conductive
polymer electrode.

WO 96/04398

PCT/GB9S/01818

An appropriate enzyme for the detection of glucose, an
analyte of particular interest, is glucose oxidase. This
enzyme is irreversibly damaged at a pH below 5 so that the
enzyme is incompatible with the conditions in which the
poly (aniline) film is grown. To produce an electrode
structure in accordance with the present invention, the
enzyme glucose oxidase is entrapped in an insulating
poly (1, 2-diaminobenzene) film which is elect rochemically
deposited on top of the poly (aniline) film. The poly(l,2-
diaminobenzene) film is chosen because it can readily be
electrochemically polymerized at a potential where-
poly (aniline) is conducting. It produces a highly active
enzyme film. The practical steps performed in order to
provide the enzyme coating .consist of transferring the
poly (aniline) -coated electrode to a solution of citric acid
and Na 2 HP0 4 solution at pH 5 containing 0.5 mol dm° Na 2 S0 4 ,
25 mmol dm" 3 1, 2-diaminobenzene, and 167 /zmol glucose
oxidase. The electrode is left in solution for six minutes
to allow adsorption of the enzyme to the poly (aniline)
surface. The poly (1, 2-diaminobenzene) film was then
deposited by holding the potential of the electrode at +0.4
V vs. SCE.for 15 minutes.

Where [Fe(CN) 6 ] 3 \is used as a mediator, the electrode
structure is placed in a stirred solution containing 3cm 3
[Fe(CN) 6 ] AT 50 mmolar in pH 5.0 buffer. The final electrode
structure is stored at room temperature in pH 5.0 solution.

Ferrocyanate ( [Fe (CN) J 30 acts as a redox mediator
shuttling charge between the enzyme and conducting polymer,
as shown schematically in Fig. 5. In its oxidized state at
+0.5 V vs. SCE and at pH 5.0, poly (aniline) is insulating.
On the addition of glucose, the film is reduced through the
following reactions:

WO 96/04398

PCI7GB95/0I818

£-D-glucose + GOx(ox) --> GOx(red) + gluconolactone

GOx(red) + 2[Fe(CN) 6 J 3 > GOx(ox) + 2[Fe(CN) 6 ] 2 -

[Fe(CN) 6 ] 2 * + PANI (ox) --> [Fe(CN) 6 ] 3 + + PAN I (red)

where GOx(ox) and GOx(red) represent the oxidized and
reduced forms of the enzyme, [Fe(CN) 6 ] 3 " and (Fe(CN)J 2 - are
the oxidized and reduced forms of the mediator, and PANI (ox)
and PANI (red) represent the oxidized, insulating state on
the one hand and a reduced and conducting state oh the other
hand of the poly (aniline) film.

It may be desirable to place the electrode in solutions
not containing glucose first to establish whether there are
any background redox reactions occurring, affecting the
conductivity of the polymer which are not directly related
to the enzyme/substrate reactions.

Tetrathiafulvalene (TTF) may also be used as a mediator
and is applied by placing the polymer coated electrode
structure in a stirred solution containing 3cm 3 pH 5.0
buffer, 50jxl of DM 50, 12jil of Triton X100 and solid TTF for
at least two hours. The final electrode structure is stored
at room temperature in TTF containing solution. TTF* and TTF
represent the oxidised and reduced forms of the mediator
respectively.

The graphs illustrated in Figs. 7 and ,8 show the
results obtained in measuring impedance of the poly (aniline)
glucose oxidase electrode structure as against time with
different concentrations of glucose in the solution. The
switching rate of the device, defined as the transient
maximum impedance gradient divided by the impedance change
over the 180s of measurement, is plotted against glucose
concentration in Fig. 8 from which it will be clear that
there is a ready relationship between the switching rate and

WO 96/0439*

PCT/GB9S/D1818

the glucose concentration. As a result, measurement of the
switching rate for any unknown glucose concentration can
readily identify the concentration.

As an alternative to the enzymatic systems it is
possible to employ bare poly (aniline) (PANI) to measure L-
ascorbic acid. Again the same AC principle was employed,
however, the fabrication of the device was much simpler
requiring only the potentiostatic growth of poly (aniline) on
an electrode. Figure 9 shows the responses to L-ascorbic
acid down to physiological rangeis and Figure 10 shows the
corresponding switching rate.

In this system, as with the others, the response to L-
ascorbic acid is concentration dependant. The system now is
much simpler as demonstrated in Figure 6.

This very simple system may be used to remove
interference caused by L-ascorbic acid. The signal produced
by ascorbic acid on a blank electrode could be used to
adjust the sensor to the actual substrate concentration. In
particular, L-ascorbic acid is a minor intereferent to the
glucose system.

In a very similar way to L-ascorbic acid, NADH can be
detected by the AC switch. Again only a bare poly (aniline)
film is required and the oxidation of NADH on poly (aniline)
shows the response of a single bare device to NADH. The
system involves the reduction of poly (aniline) film from its
insulating to conducting state.

Integration of the enzymatic response is performed
during the process as each substrate molecule reacts and
alters the redox potential and thus the impedance of the
conducting polymer. The oxidation state change remains even
if the structure is removed from the analyte solution. In
effect, the polymer film records in a memory fashion any

WO 96/04398

PCT/GB95/01818

enzymatic reactions that have taken place. Accordingly,
slow enzymatic reactions which would otherwise prove
extremely difficult to accurately measure amperometrically
can readily be detected and the concentration of the analyte
estimated using the inherent integrating property of the
conducting polymer in the single electrode device of the
present invention.

The results presented above have been obtained using an
electrode having a 20/im polymer film and have shown the
effectiveness of measurements in an AC circuit. Using the
polymer coated electrode as a single electrode, much smaller
electrodes could be used requiring only a very thin film of
polymer in the range of 0.1 to ljxm. In view of the
mechanism by which the oxidation changes in the polymer lead
to conductivity charges which can be correlated to substance
concentration, such thinner films greatly enhance the
sensitivity of the system to lower concentrations, down to
femtomolar ranges. Furthermore, the presence of a thinner
coating which does not need to bridge a gap between
electrodes increases the flexibility of the system and
extends the range of polymers which can be used.

Miniaturization of the device is possible down to
microelectrode dimensions since a large electrode area is
not required even for detection of low level analyte
concentrat ions .

Repeated electrochemical oxidation of the polymer after
each measurement can be used to reset the polymer to its
original oxidation state. This means that a single
electrode structure may be used to make a series of
measurements on the same solution. This increases accuracy
of use since the estimate of concentration can be given as
the average of numerous separate responses using the same
device .

WO 96/04398

PCT/GB95/0I818

The use of a specific redox mediator such as ferrocene
or TTF is not essential in the system. The enzyme itself
may be modified by incorporating a chemically modified
covalent redox group in the enzyme, thereby removing the -
need for the homogenous mediator.

The invention is applicable to other enzyme systems
such as horseradish peroxidase and NADH dependent enzymes.

While the invention has been described by reference to
a single polymer coated electrode, devices could be
constructed where a plurality of electrodes are formed on a
single base card, each being used independently of the
others to complete a circuit. Different polymer coatings or
enzyme systems may be applied to the different electrodes- so
that sequential AC interrogation of each electrode using the
same counter electrode will provide information for a number
of different substances or substrates in the solution.
Thus, a single device may be provide for a number of
substances using the same basic circuitry for each test.

Fig. 11 shows an example of such a device wherein two
carbon electrodes (12, 13) have coated therein a polymer
film (14, 15) which may be the same or different. A
separate enzyme system (16, 17) may be applied to each
polymer film such as horse radish peroxidase in any
insulating film and glucose oxidase in an insulating film.
Each electrode (12, 13) is connected in the AC circuit
separately to obtain a reading for the
electrode/polymer/enzyme system employed. Additional
electrodes could be provided with the same or different
polymer films and with or without an enzyme system.

WO 96/04398

PCT/GB95/01818

CLAIMS

1 A method for indicating the concentration of a
substance in solution comprising passing an alternating
voltage between a first electrode structure having coated
thereon a polymer, the polymer being in one of an oxidised
and a reduced state between which states its conductivity
varies, and a second separate counter electrode in said
solution; and measuring the change in conductivity of the
polymer coating, such measurement being representative of
the state of the polymer and thereby of the concentration of
said substance in the solution.

2 A method according to Claim 1 wherein a first
measurement is made in the absence of the substance in
solution to provide a background reading of any interfering
substances in said solution.

3 A method according to Claim 1 wherein an enzyme is
immobilised on the polymer coating, and the method is for
indicating the concentration of a substrate for said enzyme
in the solution.

4 A method according to Claim 3 wherein said first
electrode comprises a mediator associated with the polymer
or enzyme coating, for transfer of charge between the
substrate and the polymer film.

5 A method according to Claim 3 or Claim 4 wherein a
first measurement is made in the absence of said enzyme to
provide a background reading of any interfering substances
in said solution.

6 A method according to Claim 1 or 2 wherein a substrate
for an enzyme is immobilised on the polymer coating and the
method is for indicating the concentration of the enzyme in
the solution.

WO 96/04398

PCT/GB95/01818

7 A method according to any preceding claim wherein the
polymer comprises poly (aniline) .

8 A method according to any preceding claim wherein the
polymer film has a thickness in the range of 0.1 to I /im.

9 A method according to any preceding claim wherein the
first electrode structure comprises a plurality of
electrodes each using a discrete polymer coating thereon,
and an alternating voltage is passed through each electrode
separately to measure the state of the conductivity of each
polymer coating and thereby the concentration of a plurality
of substances in solution.

10 An electrode structure forming a single electrode in a
circuit for use in the method of any preceding claim
comprising an electrode having coated thereon a polymer film
the polymer being in one of an oxidised and a reduced state
between which states its conductivity varies.

11 An electrode structure according to Claim 10 comprising
an elongate electrode having an exposed face over which the
polymer is coated.

12 An electrode structure according to Claim 10 or Claim
11 wherein the polymer film is covered by a composition in
which an enzyme is immobilised.

13 An electrode according to any of Claims 10 to 12
wherein said electrode comprises a carbon microband
electrode having one or more carbon electrodes inter- layered
with insulating dielectric layers printed on a substrate,
the polymer being coated on the exposed ends of carbon of
said microband electrode.

14 An electrode according to any of Claims 10 to 13
wherein said polymer film has a thickness in the range 0.1

WO 96/04398

PCT7GB95/01818

to 1 fxm.

15 An electrode structure according to any of Claims 10 to
14 and comprising a plurality of electrodes each having
coated thereon a discrete polymer film.

16 An electrode structure according to Claim 15 wherein an
enzyme is overcoated onto one of the discrete polymer films.

WO 96/04398

PCT/GB95/D1818

SUBSTITUTE SHEET (RULE 26)

WO 96/04398

PCT/GB95/01818

FIG. 6

PANI REDUCED FROM INSULATING TO CONDUCTING

L-ASCORBIC ACID
OXIDATION PRODUCTS

SUBSTITUTE SHEET (RULE 26)

WO 96/04398

PCT/GB95/01818

A/7

Glucose Concentrations

O 1.99 mmo] dm

■ 3.98 rnmol dm

S7 5.96. mmo] dm

▼ 9.90 rnmol dm

- 3
-3
-3
-3

O
O

20 40 60 80 100 120 140 160 180

Time/s

Figure 7

SUBSTITUTE SHEET (RULE 26)

WOW/04398 PCT/GB95/01818

5/7

SUBSTITUTE SHEET (RULE ?6)

WO 96/04398

PCT/GB95/01818

6/7

Time/s

Figure 9

0.08 r- ,.

Plgore 10

SUBSTITUTE SHEET (RULE 26)

WO 96/04398

7/7

PCT7GB95/01818

FIG. 11

SUBSTITUTE SHEET (RULE 265

INTERNATIONAL SEARCH REPORT

-national Application No

PCT/GB 95/01818

A. CLASSIFICATION OF SUBJECT MATTER

IPC 6 C12Q1/00

According to tatcmaaonal Patent CUmfiotton (IPC) or to both national cUsnfication and IPC

B. FIELDS SEARCHED

Minimum documentation searched (dasstneaoon system followed by dassficanon symbols)

IPC 6 C12Q

Documentation searched other than mam mum c

) to the extent that such documents are included tn the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

C DOCUMENTS CONSIDERED TO BE RELEVANT

Category'

with

wfacrt ipfmpmtt, of the relevant p

Rdcnnt to dum No.

WO, A, 92 10584 (NEDERLANDSE ORGANISATIE
V00R T0E6EPAST NATUURWETENSCHAP 0NDERZ0EK
TNO) 25 June 1992

see page 14, line 9 - page 15, line 13;
claim 1

see page 22, line 1 - page 25, line 27

ANALYTICAL CHEMISTRY,

vol. 65, 1993 WASHINGTON DC USA,

pages 1118-1119,

P.M. BARTLETT ET AL. 'Enzyme switch
responsive to glucose 1
cited in the application
see the whole document

2-16
2-16

-/-

i are beted m the conunuaboo of bcot C

|X | Fetent farad y members aie hsted tn annex

• Special categories of ated documents
A*

r
x*

_ fee general state of the art which is not
to be of particular relevance

which may throw doubts on priority daim(*)or
itad to catahhsh the publication date of another

published after the international filing date
and not sa conflict with the application but
crted to understand the pnnopie or theory uodcrlymg the

^o^P^cuUrrehnf^^

i umnb w s step when the document a t
of parocular re le v a n c e ; the claimed invenoon
he conadajtd to involve an incentive step when the
a* ia comtaned with one or more other such docu-
toac

r of the same patent family

30 November 1995

Data of mailing of ti

01125

*essof the ISA
European Patent Office, P.B. 5S1I
NL - 2210 HV Rijswijk
Td. ( ♦ 3l-*») 340.2040, 1x 31 651
Fax 31-70) 340-3016

Van Bohemen, C

page 1 of 2

INTERNATIONAL SEARCH REPORT

• "national Application No

PCT/GB 95/01818

C^Cooomaocn) DOCUMENTS CONSIDERED TO BE RELEVANT

C*ttjorv * Quaon of document, with indication, where «ppropn»ic, of the relevant passages

Relevant to claim No.

MEDICAL & BIOLOGICAL ENGINEERING &
COMPUTING,

vol. 28, no. 3, 1 May 1990 WASHINGTON DC
USA,

pages bl0-bl7,

P.N. BARLLETT •Modified electrode surface
In amperometric biosensors. 1
see the whole document

1-16

Ferns rcTynA/Bc

•fMDMrioOlMyim)

page 2 of 2

INTERNATIONAL SEARCH REPORT

Information on patent family mcmbai

' -national Application No

PCT/GB 95/01818

Patent document
cited in search report

Publication
date

Patent famUy
member^*)

Publication
dale

W0-A-9210584

25-06-92

NL-A-

9002764

01-07-92

0E-D-

69104496

10-11-94

DE-T-

69104496

23-02-95

EP-A-

0561966

29-09-93

US-A-

5422246

06-06-95

Pom tCTASA/St

Ml? MM) <j»* im)

Internet Archive Audio

Featured

Top

Images

Featured

Top

Software

Featured

Top

Books

Featured

Top

Video

Featured

Top

Mobile Apps

Browser Extensions

Archive-It Subscription

Save Page Now

Full text of "USPTO Patents Application 10687850"

See other formats